Conventional drug discovery and development takes an average of 12 years and costs hundreds of millions of dollars for each new drug that reaches patients. Repositioning of approved drugs and clinical-stage compounds with existing preclinical and clinical data can greatly expedite the process, particularly for rare, low-prevalence diseases and for rapidly-spreading infectious diseases, such as Zika virus. We employed new technologies and approaches for screening, such as phenotypic cell-based disease models using human-derived induced pluripotent stem (iPS) cells and high-content screening platforms. Our approach is to collaborate with leading investigators from across the research ecosystem, including at NIH, academic institutions, and biopharmaceutical companies. Our objectives include (1) development of disease relevant assays using human neural progenitor cells and astrocytes; (2) drug repurposing screening to identify active compounds that protect neuronal cells from Zika virus caused cell death and inhibit virus replication; (3) confirmation of compound activity in in vitro assays and animal models; and (4) advancement of the newly identified compounds to clinical trials for the treatment of Zika virus infection. Despite rapid progress in the preclinical development of vaccines against Zika virus (ZIKV), testing the safety and efficacy of vaccines in humans can take a substantial amount of time. Effective countermeasures, including small-molecule therapeutics, are also urgently needed. In response to the current global health emergency posed by the ZIKV outbreak and its link to microcephaly and other neurological conditions, we continued our drug discovery and development research for treatment of ZIKV infections. We developed a new ZIKV NS1 protein assay and optimized it in 1536-well plate format for high throughput compound screening. We performed a drug repurposing screen of a library of 6016 compounds, including approved drugs, clinical trial drug candidates and other pharmacologically active compounds. To confirm primary screening hits, we designed and applied a set of secondary assays including the ATP content cell viability assay and ZIKV E-protein assay. We confirmed 134 compounds with selective inhibitory activity against ZIKV infection in vitro. We also collaborated with Drs. Alexey Terskikh (Sanford Burnham Prebys Medical Discovery Institute) and Hengli Tang (Florida State University) for testing our top lead compounds in two ZIKV mouse models. The results confirmed two compounds with potent activity suppressing ZIKV replication in the animal models. We are studying the mechanism of action for these compounds. We worked with Dr. Hengli Tang lab for development of a semi-automated ZIKV titer. The virus titer assay is a good standard experiment for testing antiviral effects of vaccines and drugs, which quantitates infectious virion production. The traditional virus titer assay is labor-intensive, low-throughput, and cannot meet the need for drug development. We have applied the use of an electronic multichannel pipette and computational algorithms for carrying out semi-automated experiments and data analysis. This new ZIKV titer assay improved the compound screening throughput by 50-to-100-fold and allowed us to evaluate anti-ZIKV compounds quickly. This new method can be broadly applied to assaying titer for other viruses. In addition, we conducted siRNA screening with the druggable genome collection, identifying a set of genes involved in ZIKV replication in human cells. These results were incorporated with the screening data of the interaction between ZIKV and human proteins, as well as drug repurposing screening data. These datasets are being used for a systematic analysis to interrogate potential mechanisms of pathogenesis and to identify new drug targets. Upon publication, all data will be deposited in PubChem to allow open access to other researchers.